TC-1 Tumor-bearing Mice Research Articles

Synergy Effects of HPV E6-E7 Encoding mRNA and Nucleic Acid Immunostimulators Improve Therapeutic Potential in TC-1 Graft Tumor.

Cervical cancer is the second most common cancer among women globally and the most prevalent cancer in developing countries, which was caused by human papillomavirus (HPV) infection. Messenger RNA (mRNA) vaccines have opened up new avenues for vaccine development and pandemic preparedness with potent scalability, which may possess the potential antitumor effects of an mRNA-HPV therapeutic vaccine containing nononcogenic E6 and E7 proteins. Here, we reported a lipid nanoparticle (LNP) plus nucleic acid immunostimulators (CPG 1018 and Poly I:C) mRNA vaccine platform. The LNP-CPG 1018 capsulated HPV E6-E7 mRNA significantly promoted the maturation of bone marrow-derived dendritic cells (BMDC) in vitro and were capable of efficiently migrating to lymph nodes (LN) in vivo. In TC-1 tumor-bearing mice, the subcutaneous immunization of LNP-CPG 1018 capsulated HPV E6-E7 mRNA elicited robust tumor-specific T-cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In conclusion, the LNP-CPG 1018 system is a promising delivery platform for facilitating the development of HPV E6-E7 mRNA cancer vaccines.

Abstract 4103: Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo

Abstract Human Papillomavirus (HPV), particularly HPV16, is a known causative agent of cancers in various anatomical sites, notably the anogenital and oropharyngeal regions. Despite the availability of prophylactic HPV vaccines for almost two decades, vaccination rates among young females, who are at a heightened risk for cervical intraepithelial neoplasia (CIN) and subsequent invasive cancer, remain suboptimal. The current standard of care for cervical cancer, involving invasive surgery and chemotherapy-based treatments, underscores the pressing need for safer and more effective therapeutics to enhance patient survival and quality of life. In this context, while several HPV16-E6/E7 targeted therapeutic vaccines are in development and clinical trials, their clinical efficacy has been limited due to inadequate antigen presentation and suboptimal immunogenicity. Addressing this, our team has developed a novel, highly immunogenic mRNA therapeutic cancer vaccine, engineered to activate a robust T cell-mediated adaptive immune response and exhibit strong anti-tumor potential. Our innovative approach includes the design of an HPV16-E6/E7 mRNA vaccine (ABO2101) that incorporates a unique antigen-expressing cassette. This cassette features endolysosomal trafficking domains and antigen presenting cell (APC) activation stimulators, enhancing both Class I and Class II antigen presentation and APC maturation, thereby amplifying antigen-specific T cell responses. In both murine and human-derived experimental models, ABO2101 has demonstrated a significantly more potent adaptive T cell response against HPV16 antigens compared to other HPV16 mRNA therapeutic vaccines. In antitumor efficacy evaluations using TC-1 tumor-bearing mice, ABO2101 single-dose administration successfully eradicated both early-stage (50 mm³) and advanced (500 mm³) tumors. This was accompanied by a marked increase in HPV16-specific CD8+ cytotoxic T lymphocytes within the tumor microenvironment. Moreover, in prophylactic and adjuvant therapy models, ABO2101 prevented tumor growth across various dosages, indicating its robust preventative capabilities. Further, the combination of ABO2101 with immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, displayed a synergistic effect in both immune-oncology sensitive and resistant animal models, suggesting a potential for combinational clinical therapy. In conclusion, our development of ABO2101, a mRNA-based HPV16-E6/E7 therapeutic cancer vaccine inclusive of APC maturation signals, represents a groundbreaking advancement in HPV-associated cancer treatment, as supported by our compelling preclinical data. Citation Format: Liang Du, Hongya Han, Jingshu Ma, Zhenxing Yang, Jinjuan Mao, Jijun Yuan, Bo Ying. Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4103.

Peptide-Based Therapeutic HPV Cancer Vaccine Synthesized via Bacterial Outer Membrane Vesicles.

Peptide-based vaccines have broad application prospects because of their safety, simple preparation, and effectiveness, especially in the development of personalized cancer vaccines, which have shown great advantages. However, the current peptide-based vaccines often require artificial synthesis and intricate delivery technology, which increases the cost and complexity of preparation. Here, we developed a simple technique for combining a peptide and a delivery system using the natural secretion system of bacteria. Specifically, we biosynthesized an antigenic peptide in bacteria, which was then extracellularly released through the bacterial secretory vesicles, thus simultaneously achieving the biosynthesis and delivery of the peptide. The system utilizes the natural properties of bacterial vesicles to promote antigen uptake and dendritic cell (DC) maturation. Therefore, tumor-specific CD4+ Th1 and CD8+ cytotoxic T lymphocyte (CTL) responses were induced in TC-1 tumor-bearing mice, thereby efficiently suppressing tumor growth. This research promotes innovation and extends the application of peptide-based vaccine biosynthesis technology. Importantly, it provides a new method for personalized cancer immunotherapy that uses screened peptides as antigens in the future.

Engineered Norovirus-Derived Nanoparticles as a Plug-and-Play Cancer Vaccine Platform.

In recent years, virus-derived self-assembled protein nanoparticles (NPs) have emerged as attractive antigen delivery platforms for developing both preventive and therapeutic vaccines. In this study, we exploited the genetically engineered Norovirus S domain (Nov-S) with SpyCatcher003 fused to the C-terminus to develop a robust, modular, and versatile NP-based carrier platform (Nov-S-Catcher003). The NPs can be conveniently armed in a plug-and-play pattern with SpyTag003-linked antigens. Nov-S-Catcher003 was efficiently expressed in Escherichia coli and self-assembled into highly uniform NPs with a purified protein yield of 97.8 mg/L. The NPs presented high stability at different maintained temperatures and after undergoing differing numbers of freeze-thaw cycles. Tumor vaccine candidates were easily obtained by modifying Nov-S-Catcher003 NPs with SpyTag003-linked tumor antigens. Nov-S-Catcher003-antigen NPs significantly promoted the maturation of bone marrow-derived dendritic cells in vitro and were capable of efficiently migrating to lymph nodes in vivo. In TC-1 and B16F10 tumor-bearing mice, the subcutaneous immunization of NPs elicited robust tumor-specific T-cell immunity, reshaped the tumor microenvironment, and inhibited tumor growth. In the TC-1 model, the NPs even completely abolished established tumors. In conclusion, the Nov-S-Catcher003 system is a promising delivery platform for facilitating the development of NP-based cancer vaccines.

Abstract LB566: Class 1 HDAC inhibition induces antitumor immunity by NF-kB-mediated enhanced metabolic fitness and generation of unique effector function enriched memory CD8 T cell subtype

Abstract Histone/protein deacetylases (HDACs) are a class of enzymes that regulate gene expression and are involved in regulating the transcription process as well as regulating fundamental cellular processes such as proliferation, differentiation and development. Class I HDACs (which includes HDAC1, 2, 3, and 8) contribute in epigenetic regulations of T cell tolerance. Under anergic T cell condition, HDAC1 and HDAC2 are involved in the histone deacetylation. However, the role of HDACs in CD8 T cell function and differentiation is not well understood. Therefore, we aimed to understand the effect of HDAC inhibition (HDACi) on CD8 T cells phenotype and functionality by using Entinostat (ENT), a class I HDAC (HDAC1 and 3) inhibitor. We found that ENT induces strong immune-mediated anti-tumor effects in TC-1 and B16 tumor-bearing mice. Mechanistically, ENT modulated the tumor microenvironment through an increased infiltration of CD8 T cells, enhanced CD8 T cell antigen-specificity and CD8 T cell functionality represented by interferon gamma (IFN-γ) and granzyme B (GrB) secretion. Our in vitro and in vivo data also indicate that ENT enhances central memory phenotype (CD62L+, CD44+) of CD8 T cells. We also found that ENT-treated CD8 T cells have distinct metabolic phenotype compared to activated CD8 T cells. Specifically, these cells relied more on amino acid metabolism while, unexpectedly, they had lower fatty acid uptake with enhanced mitochondrial potential (TMRM). Interestingly, ENT-induced central memory CD8 T cells also showed increased mitochondrial potential as well as reliance on amino acid metabolism. Therefore, ENT treatment induced memory cells with a unique phenotype different from the classical memory CD8 T cells with enhanced effector functions and reliance on distinct metabolic pathways. Furthermore, ENT-treated CD8 T cells have higher peaks at H3K27Ac site compared to the control. Since these peaks are mostly associated with genes such as IFN-γ, GrB, Pim1, EOMES, and IL-2, the known canonical target genes of NF-κB in CD8 T cells, we further dissected the role of NF-κB in these ENT-mediated effects. First, motif analysis showed that NF-κB binding motif is enriched in differential H3K27Ac sites associated with HDACi. Next, flow cytometry and immunofluorescence analysis showed that although ENT does not increase total NF-κB levels in CD8 T cells, it enhances its binding to the chromatin due to enhanced gene accessibility. This research highlights the significance of epigenetic modulation in enhancing the immune-mediated anti-tumor effects and provide fundamental understanding of the mechanism of action of HDAC inhibition in CD8 T cells. Citation Format: Nour Shobaki, Pankaj Gaur, Rahul Nadre, Vivek Verma, Peter Ordentlich, Lei Wang, Nazli Jafarzadeh, Wael Traboulsi, Mikayel Mkrtichyan, Seema Gupta, Samir N. Khleif. Class 1 HDAC inhibition induces antitumor immunity by NF-kB-mediated enhanced metabolic fitness and generation of unique effector function enriched memory CD8 T cell subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB566.

Histone Deacetylase as a Valuable Predictive Biomarker and Therapeutic Target in Immunotherapy for Non-Small Cell Lung Cancer.

PurposeHistone deacetylase inhibitors (HDACis) are epigenetic regulators and used clinically for hematopoietic malignancies. Recently, HDACis have received attention as a factor that modulates the immune system. In this study, the role of histone deacetylase (HDAC) expression as a predictive marker in lung cancer patients who were treated with immune checkpoint inhibitors (ICIs) and the role of HDACi and ICI combination treatment in the mouse tumor model were analyzed.Materials and MethodsThe overall response rate (ORR) and progression-free survival (PFS) were analyzed by the expression of HDAC. In vitro assay, the mRNA and protein expression levels of cytokines and programmed death-ligand 1 (PD-L1) were analyzed after HDACi treatment. In vivo assay, TC-1 tumor-bearing mice were treated with HDACi and mouse programmed cell death 1 (PD-1) inhibitor.ResultsThe HDAC6 low expression group showed high ORR and prolonged PFS. When the selective HDAC6 inhibitor was administered to the A549 cell line, the levels of interleukin-1β and interleukin-6 decreased and the expression of PD-L1 was reduced. Mice that received both the mouse PD-1 inhibitor and pan-HDACi had a smaller tumor size than that of the mice from the control group. Moreover, mice treated with the mouse PD-1 inhibitor and pan-HDACi generated greater numbers of E7-specific CD8+ T cells.ConclusionHDAC6 expression can predict the prognosis of non–small cell lung cancer patients who were treated with ICIs. Furthermore, co-treatment with HDACi and PD-1 inhibitor was shown to decrease the tumor growth rate and create a favorable tumor microenvironment for cytotoxic T lymphocytes in the TC-1 mouse model.

A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses.

Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

Tumor targeting nanoparticle E749-57-HSP110-RGD elicits potent anti-tumor immune response in a CD8-dependent manner in cervical cancer-bearing mouse model

ABSTRACT Our previous research verified that HSP (heat shock protein) 110 could enhance the anti-tumor effect of HPV16 E749-57 epitope. In this study, to optimize the immunotherapy of this vaccine type, we developed and evaluated the anti-tumor immunity of a nanoparticle vaccine format assembling with E749-57-HSP110 fusion expression plasmid and RGD-GGG-K18 polypeptide. The nanoparticle vaccine was self-assembled from positively charged RGD-GGG-K18 polypeptide and negatively charged fusion expression plasmid pIRES2-3× E7-HSP110-EGFP. The particle size, stability, expression of E749-57-HSP110 fusion protein and the target ability of nanoparticle were determined, respectively. Specific CTL responses were determined by E7 tetramer staining and cytotoxicity assay in TC-1 tumor-bearing mice (CD4/CD8 knockout). The preventive and therapeutic experiments of nanoparticle vaccine were investigated in TC-1 tumor-bearing mice. Results showed that the RGD-GGG-K18 polypeptide and pIRES2-3× E7-HSP110-EGFP plasmid self-assembled nanoparticles about 100 nanometers in diameter when the charge ratios of peptide/plasmid were 2. The nanoparticles effectively entered TC-1 cells directed by RGD target-peptide, and correctly expressed the E7-HSP110 fusion protein. The HSP110 effectively facilitated nanoparticles activating CD8+T cells than nanoparticles without HSP110, including the CD8+ T cell number and the IFN-γ level; in contrast, the CD4+T cells immune response remained indiscriminate among the mice groups. This nanoparticle formulation inhibited tumor growth and prolonged the survival duration in the prophylactic and therapeutic mouse models. Therefore, the RGD-based tumor-targeting nanoparticle expressing E749-57-HSP110 fusion protein can efficiently evoke anti-tumor activity and thus suggests it might be a favorable candidate for cervical cancer immunotherapy.

P53.04 KUgliptin, a Novel and Potent DPP4 Inhibitor, Improves of Combined Therapy with Immune Checkpoint Inhibitor (ICI) in Lung Cancer Immunotherapy

Lung cancer is the most common cause of death rate among all cancer related diseases. Although several drugs including immunotherapeutics (Immune checkpoint inhibitor; ICI) have been developed and used in recent years, the response rate is limited to about 25%. Therefore, many researchers have tried to combine treatment with other drugs to increase the therapeutic efficiency of ICI. Dipeptidyl peptidase 4 (DPP4) is a ubiquitously expressed transmembrane exopeptidase on the cell surface of many hematopoietic cells. According to some reports, DPP4 inhibitors have the ability to suppress tumor proliferation and tumor metastasis. KUgt-123, is a novel and potent DPP4 inhibitor, it is chemical structural based in adamantane structure modified. This study was performed to combined treatment of KUgt-123 for improvement of therapeutic efficacy of immune checkpoint inhibitor for lung cancer. To confirm the cytotoxicity effect of KUgt-123, it was dose dependently treated in human lung carcinoma epithelial cell line (A549) and mouse Lewis lung carcinoma (LLC) cells then using MTT assay. PD-L1 expression level was determined by western blot analysis. TC-1 tumor-bearing mice were treated with either PD-1 antibody treatment group and/or KUgt-123 treatment. KUgt-123 was shown to inhibit the cell proliferation in A549 cells and LLC cells. The result that cell growth was reduced dose dependently of KUgt-123. PD-L1 expression was reduced by KUgt-123 treatment in TC-1 cells. TC-1 tumor bearing C57BL/6 mice, the size of tumors was reduced by alone KUgt-123 treatment and significantly reduced co-treatment with mouse PD-1 antibody. Our data provide evidence that KUgt-123 might be a good co-treatment reagent for improves the efficacy of ICI in lung cancer treatment.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Endoplasmic reticulum stress enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV vaccines

BackgroundEndoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines.MethodsIn the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 μg/mouse).ResultsTreatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice.ConclusionsOur data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.

Abstract 720: Anti-OX-40 agonist antibody significantly increases therapeutic efficacy of TVGV-1 vaccine for HPV-positive cancers

Abstract Background: Human papillomavirus 16 (HPV16) E7 protein is a potential target antigen for HPV-positive cancers due to its interaction with pRb tumor suppressor protein. The clinical-stage therapeutic vaccine, TVGV-1, comprises of the PE-E7-K3 fusion protein and the adjuvant GPI-0100, has shown therapeutic effect preclinically by stimulating T cell response targeting HPV16 E7 protein. TVGV-1 was well tolerated clinically in more than 30 HPV+ cervical intraepithelial neoplasia (CIN) patients. Agonistic antibodies targeting OX40 (CD134) have shown remarkable single agent anti-tumor effect by modulating T cell response surrounding tumor environment, as well as the ability to combine with other immunotherapies in several mouse tumor models. Here we evaluated the synergistic therapeutic potential of TVGV-1 vaccine combined with anti-OX-40 agonist antibody for HPV-positive cancer therapy. Methods: Therapeutic and immune efficacy of TVGV-1 in combination with anti-OX-40 agonist antibody was tested in E7 antigen expressing TC-1 tumor mouse model. Mice with various treatments were assessed for tumor growth and survival. Peripheral and tumor-infiltrating immune cell profiles with various treatments were also assessed by flow cytometry. Results: Our results revealed that both the HPV16 E7-specific humoral and cell-mediated immunities solely elicited by TVGV-1 vaccination was synergistically enhanced more than two-fold by concomitant anti-OX-40 agonist antibody treatment. In a TC-1 tumor model, tumor growth and overall survival was not protected by OX-40 antibody treatment alone, and mice was protected with prolonged survival benefit when treated in combination of TVGV-1 and OX-40 antibody. In TC-1 tumor bearing mice, about 10% decrease of peripheral CD3+ T cell was observed upon TC-1 tumor development, and restoration was only observed when mice were treated with combined TVGV-1 and OX-40 antibody. When tumor infiltrating lymphocytes recognizing HPV E7 antigen were prepared from these mice, the amount of tumor-infiltrating mononuclear cells and CD3+ T cells were dramatically increased with combination treatment when compared to control group or to treatment alone groups. Conclusions: We demonstrated efficacy with combination approach between TVGV-1 vaccine and anti-OX-40 agonistic antibody in a pre-clinically mouse model. This enhanced activity is likely due to modulation of T-cell population in tumor and in periphery. This result will guide to a better design to perform clinical studies in order to obtain better outcome with TVGV-1 cancer vaccine intended for treating HPV E7 bearing tumors. Citation Format: Yin-Ching Lin, Yi-Tsui Chiu, Yi-Chia Lin, Jiun-Ming Wu, Chien-Hung Chen, Wen-Fang Cheng, Chia-Mao Wu. Anti-OX-40 agonist antibody significantly increases therapeutic efficacy of TVGV-1 vaccine for HPV-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 720.

TP-0184 Inhibits ALK2/ACVR1, Decreases Hepcidin Levels, and Demonstrates Activity in Preclinical Mouse Models of Functional Iron Deficiency

BackgroundHepcidin is a liver peptide hormone that functions as a master regulator of bioavailable iron. Hepcidin levels become constitutively high in chronic inflammatory conditions where functional iron deficiency (FID) becomes a challenge. Approaches to target hepcidin expression or function have emerged as attractive strategies to reverse the complications of functional iron deficiency. Targeting the activation of the ALK2/ACVR1 receptor has been validated as a promising approach to target hepcidin. Activation of ALK2/ACVR1 in the liver induces the SMAD-driven transcription of hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Current therapeutic approaches for anemia of chronic disease (ACD) rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which adequately address the underlying pathological deficit of functionally low iron levels. TP-0184 has been developed as a potent and selective inhibitor of ALK2/ACVR1, and it was hypothesized that targeting of ALK2/ACVR1 with TP-0184 would reverse the effects of FID in preclinical models of anemia. TP-0184 is a small-molecule inhibitor of ALK2 kinase activity, exhibiting an IC50 of 5 nM in biochemical assays.AimsThe current study seeks to establish proof-of-concept that TP-0184 targets ALK2/ACVR1 and reverses the effects of FID. Utilizing the unique experience in a single patient IND, the clinical effects of TP-0184 on serum hepcidin levels was evaluated.MethodsMultiple preclinical models of anemia were used to evaluate the efficacy of TP-0184. These models included: a short-term chemical induced anemia by turpentine oil (TO), a model of anemia of cancer using the TC-1 syngenic lung cancer cell line, and an infectious disease model using heat-inactivated Brucella abortus (HKBA).ResultsTP-0184 has demonstrated consistent preclinical activity in three mouse efficacy models of FID. In the first model, TP-0184 reversed hepcidin induction more than 6.8-fold in mice induced with TO. The simplicity of the TO model has allowed the exploration of numerous dose levels and treatment schedules of TP-0184 and the ability to correlate these with pharmacokinetic plasma levels of the compound. In the second model, TP-0184 reversed elevated hepcidin levels and increased iron levels induced in TC-1 tumor bearing mice. Hepcidin was reduced nearly 3-fold with doses as low as 25 mg/kg. In the third model, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection of HKBA. In addition to these preclinical models, a single cancer patient received oral administration of TP-0184 in an emergency setting. Although the primary objectives of the single patient study did not include anemia, pharmacokinetic and pharmacodynamic measurements related to anemia were measured in the patient. The patient was administered 2 cycles of TP-0184, where each cycle evaluated a different formulation. Before the first cycle, the patient had a baseline serum hepcidin level of 559 pg/mL. These levels dropped to below the detectable limit within 24 hours of the first dose with TP-0184. Hepcidin levels remained undetectable for the duration of TP-0184 treatment. After a break in treatment, the patient was administered a second cycle of TP-0184 in an alternative formulation. During this cycle, the patient's hepcidin levels decreased from 182 pg/mL to 69 pg/mL after 24 hours of the first dose (cycle 2). With daily dosing, the patient's serum hepcidin levels continued to decrease to undetectable levels by at least 9 days. Importantly, serum iron levels mirrored serum hepcidin levels well. Plasma pharmacokinetics were also obtained in this patient.ConclusionTargeting ALK2/ACVR1 is an attractive strategy to downregulate hepcidin levels and reverse the effects of FID. TP-0184, a potent ALK2/ACVR1 inhibitor, has demonstrated consistent activity in multiple preclinical models and now has preliminary proof-of-concept evidence that it can modulate hepcidin levels in man. The development of TP-0184 is currently progressing through preclinical development and a Phase I first-in-human study focusing on FID is currently being planned. DisclosuresPeterson:Tolero Pharmaceuticals, Inc.: Employment. K:Tolero Pharmaceuticals, Inc.: Employment. Whatcott:Tolero Pharmaceuticals, Inc.: Employment. Siddiqui-Jain:Tolero Pharmaceuticals, Inc.: Employment. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Bearss:Tolero Pharmaceuticals, Inc.: Employment. Warner:Tolero Pharmaceuticals, Inc.: Employment.

Enhancement of therapeutic DNA vaccine potency by melatonin through inhibiting VEGF expression and induction of antitumor immunity mediated by CD8+ T cells.

To be effective, therapeutic cancer vaccines should stimulate both an effective cell-mediated and a robust cytotoxic CD8+T-cell response against human papillomavirus (HPV)-infected cells to treat the pre-existing tumors and prevent potential future tumors. In this study, the therapeutic experiments were designed in order to evaluate antitumor effect against the syngeneic TC-1 tumor model. The anti-tumorefficacy of a HPV-16 E7 DNA vaccine adjuvanted with melatonin (MLT) was evaluated in a C57BL/6 mousetumormodel by measuringtumorgrowth post vaccination and the survival rate oftumor-bearingmice, analyzing the specific lymphocyte proliferation responses in control and vaccinatedmiceby MTT assay. TheE7-specific cytotoxic T cells (CTL) were analyzed bylymphocyteproliferation and lactate dehydrogenates (LDH) release assays. IFN-γ, IL-4 and TNF-α secretion in splenocyte cultures as well as vascular endothelial growth factor (VEGF) and IL-10 in the tumor microenvironment were assayed by ELISA. Our results demonstrated that subcutaneous administration of C57BL/6micewith a DNA vaccine adjuvanted with MLT dose-dependently and significantly induced strong HPV16 E7-specific CD8+ cytotoxicity and IFN-γ and TNF-α responses capable of reducing HPV-16 E7-expressing tumor volume. A significantly higher level of E7-specific T-cell proliferation was also found in the adjuvanted vaccine group. Furthermore, tumor growth was significantly inhibited when the DNA vaccine was combined with MLT and the survival time of TC-1 tumor bearing mice was also significantly prolonged. In vivo studies further demonstrated that MLT decreased the accumulation of IL-10 and VEGF in the tumor microenvironment of vaccinated mice. These data indicate that melatonin as an adjuvant augmented the cancer vaccine efficiency against HPV-associated tumors in a dose dependent manner.

Coinjection of IL2 DNA enhances E7-specific antitumor immunity elicited by intravaginal therapeutic HPV DNA vaccination with electroporation.

The generation and use of therapeutic human papillomavirus (HPV) DNA vaccines represent an appealing treatment method against HPV-associated cervical cancer due to their safety and durability. Previously, we created a therapeutic HPV DNA vaccine candidate by linking the HPV16-E7 DNA sequence to calreticulin (CRT/E7), which we showed could generate significant E7-specific cytotoxic T lymphocyte (CTL)-mediated anti-tumor immune responses against HPV16 oncogenes expressing murine tumor model TC-1. Here we assess the therapeutic efficacy of intravaginal immunization with pcDNA3-CRT/E7 followed by electroporation. In addition, we examined whether coadministration of DNA encoding IL2 with the pcDNA3-CRT/E7 could improve the T cell responses elicited by pcDNA3-CRT/E7. TC-1 tumor-bearing mice vaccinated intravaginally with both pcDNA3-CRT/E7 and IL2 DNA followed by electroporation induced stronger local anti-tumor CTL response in comparison to mice that received other treatment regimens. Additionally, we found that coadministration of IL2 DNA with pcDNA3-CRT/E7 modified the tumor microenvironment by decreasing the population of regulatory T cells and myeloid derived suppressor cells relative to that of CTLs. Our data demonstrates the translational potential of local administration of IL2 and pcDNA3-CRT/E7 followed by electroporation in treating cervicovaginal tumors.

The ALK-2 Inhibitor, TP-0184, Demonstrates High Distribution to the Liver Contributing to Significant Preclinical Efficacy in Mouse Models of Anemia of Chronic Disease

In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD. DisclosuresPeterson:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

In vivo stepwise immunomodulation using chitosan nanoparticles as a platform nanotechnology for cancer immunotherapy.

Dentritic cell (DC)-based cancer immunotherapy faces challenges in both efficacy and practicality. However, DC-based vaccination requires multiple injections and elaborates ex vivo manipulation, which substantially limits their use. Therefore, we sought to develop a chitosan nanoparticle (CH-NP)-based platform for the next generation of vaccines to bypass the ex vivo manipulation and induce immune responses via active delivery of polyinosinic-polycytidylic acid sodium salt (poly I:C) to target Toll-like receptor 3 (TLR3) in endosomes. We developed CH-NPs encapsulating ovalbumin (OVA) as a model antigen and poly I:C as the adjuvant in an ionic complex. These CH-NPs showed increased in vivo intracellular delivery to the DCs in comparison with controls after injection into tumor-bearing mice, and promoted DC maturation, leading to emergence of antigen-specific cytotoxic CD8+ T cells. Finally, the CH-NPs showed significantly greater antitumor efficacy in EG.7 and TC-1 tumor-bearing mice compared to the control (p < 0.01). Taken together, these data show that the CH-NP platform can be used as an immune response modulatory vaccine for active cancer immunotherapy without ex vivo manipulation, thus resulting in increased anticancer efficacy.

Toll-like receptor 3-induced immune response by poly(d,l-lactide-co-glycolide) nanoparticles for dendritic cell-based cancer immunotherapy.

Dendritic cells (DCs) are potent professional antigen-presenting cells that are capable of initiating a primary immune response and activating T cells, and they play a pivotal role in the immune responses of the host to cancer. Prior to antigen presentation, efficient antigen and adjuvant uptake by DCs is necessary to induce their maturation and cytokine generation. Nanoparticles (NPs) are capable of intracellular delivery of both antigen and adjuvant to DCs. Here, we developed an advanced poly(d,l-lactide-co-glycolide) (PLGA)-NP encapsulating both ovalbumin (OVA) as a model antigen and polyinosinic-polycytidylic acid sodium salt (Toll-like receptor 3 ligand) as an adjuvant to increase intracellular delivery and promote DC maturation. The PLGA-NPs were taken up by DCs, and their uptake greatly facilitated major histocompatibility class I antigen presentation in vitro. Moreover, vaccination with PLGA-NP-treated DCs led to the generation of ovalbumin-specific CD8+ T cells, and the resulting antitumor efficacy was significantly increased in EG.7 and TC-1 tumor-bearing mice compared to control mice (P<0.01). Taken together, these findings demonstrated that the PLGA-NP platform may be an effective method for delivering tumor-specific antigens or adjuvants to DCs.

Adjuvant effect of docetaxel on HPV16 L2E6E7 fusion protein vaccine in a mouse model

We previously demonstrated that the antineoplastic agent docetaxel enhanced the immune response to an influenza vaccine. This study evaluated the adjuvant effect of docetaxel (DOC) on the therapeutic efficacy of HPV16 L2E6E7 fusion protein (HPV-LFP) in mice inoculated with TC-1 cells. The results demonstrated that docetaxel significantly enhanced the therapeutic effect of HPV-LFP on TC-1 cell-induced tumors in mice. The injection of HPV-LFP in combination with docetaxel in TC-1 tumor-bearing mice significantly reduced tumor volume and weight, and a greater percent survival was detected than mice treated with HPV-LFP alone. The inhibition of tumors was associated with significantly increased serum antigen-specific IgG and isotypes, activated CTLs, increased IFN-γ-secreting T cells, and decreased Treg cells and IL-10-secreting cells in spleen. In addition, down-regulation of IL-10, VEGF and STAT3, up-regulation of IFN-γ and decreased Treg cells in the tumor microenvironment may also important contributing factors to the antitumor effect. It may be valuable to use a DOC-containing water to dilute HPV-LFP powder before injection in patients because of its excellent adjuvant effect on HPV-LFP and solubility in water.